Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (18): 3556-3573.doi: 10.3864/j.issn.0578-1752.2023.18.006

• PLANT PROTECTION • Previous Articles     Next Articles

Fluorescent Labeling and Observation of Infection Structure of Fusarium verticillioides

HA DanDan1(), ZHENG HongXia2(), ZHANG ZhenHao2, ZHU LiHong3, LIU Hao1, WANG JiaoYu2,3(), ZHOU Lei2()   

  1. 1 College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457
    2 Institute of Agro-Product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences/State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Hangzhou 310021
    3 Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
  • Received:2023-04-25 Accepted:2023-07-04 Online:2023-09-16 Published:2023-09-21
  • Contact: WANG JiaoYu, ZHOU Lei

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Abstract:

【Objective】Taking the dominant pathogen Fusarium verticillioides causing maize ear rot and producing fumonisin as the research object, the objective of this study is to construct fluorescent strains that constitutively express green fluorescent protein GFP, red fluorescent protein DsRED, peroxisome targeted marker protein DsRED-PTS1 and F-actin cytoskeleton binding protein LifeAct-GFP, observe the penetration structure of F. verticillioides infecting maize silk epidermis, and to clarify the regulatory mechanisms and influencing factors regulating the infection structure formation of F. verticillioides.【Method】Fluorescent expression vectors pCOM-GFP, pCOM-DsRED, pCOM-DsRED-PTS1, and pCOM-LifeAct-GFP were transformed into the wild-type strain D85-2 of F. verticillioides through Agrobacterium-mediated genetic transformation. The transformants containing the target genes were identified by PCR, and the expression and distribution of various fluorescent proteins were observed under laser confocal microscopy. Maize was inoculated to observe the infection structure. The penetration structure and dynamic assembly of F-actin were observed by simulating the plant epidermal penetration experiment with cellophane membrane. The influencing factors of penetration structure formation were detected using F-actin polymerization inhibitor Latruculin A, tricyclazole, and reactive oxygen species (ROS) inhibitor DPI (diphenyleneiodonium chloride).【Result】A genetic transformation method using geneticin G418 as a resistance marker was established. FV-GFP and FV-DsRED strains have clear and bright green fluorescence and red fluorescence, respectively. The red fluorescence in FV-DsRED-PTS1 strain is distributed as round dots in conidia and hypha, which is consistent with the distribution characteristics of peroxisome in fungi. FV-LifeAct-GFP strain has filamentous green fluorescence in conidia and hypha, which is consistent with the distribution characteristics of F-actin protein in fungi. During the infection process of maize silk epidermis, an infection structure resembling a hyphopodium was observed at the top of the hyphae. The ring-forming assembly of F-actin protein in the penetration structure was observed in the mimic penetration experiment on the cellophane membrane. Drug stress experiments have shown that the formation of penetrating structures is regulated by F-actin protein polymerization and ROS. Meanwhile, tricyclazole can inhibit penetration.【Conclusion】Four fluorescent labeled strains were constructed, with clear cell localization and normal growth phenotype and pathogenicity, which can meet the research needs related to pathogenic mechanisms. F. verticillioides can form a hyphopodium like infection structure with swollen hyphae at the top when infecting maize. It is confirmed that F-actin cyclization and polymerization, cytoskeleton assembly and ROS regulation are the key factors affecting the formation of infection structure of F. verticillioides.

Key words: Fusarium verticillioides, fluorescent labeling, peroxisome, infection structure, F-actin, reactive oxygen species (ROS)

Fig. 1

Construction of fluorescent labeling vectors"

Table 1

Primers used in this study"

引物名称
Primer name		 序列
Sequence (5′-3′)		 产物长度
Product length (bp)
G418-F GTGCCCTGAATGAACTGC 517
G418-R TCACGGGTAGCCAACG
GFP-F CTGGACGGCGACGTAAACGG 564
GFP-R GGACTGGGTGCTCAGGTAGTGG
RED-F ACGGCCACGAGTTCGA 549
RED-R CGTTGTGGGAGGTGATGT

Fig. 2

Sensitivity test of F. verticillioides wild-type strain D85-2 to geneticin G418"

Fig. 3

Confirmation of the F. verticillioides fluorescent transformants using PCR amplification"

Fig. 4

Colony morphology, growth rate, conidiation and pathogenicity of the four fluorescent transformants"

Fig. 5

Observation of GFP and DsRed fluorescence in the FV-GFP and FV-DsRED transformants"

Fig. 6

Observation of F-actin cytoskeleton with GFP fluorescence and peroxisomes with DsRED fluorescence in the FV-LifeAct- GFP and FV-DsRED-PTS1 transformants, respectively"

Fig. 7

Observation of GFP and DsRED fluorescence mixed with the cell wall staining agent CFW in the FV-GFP and FV-DsRED strains, respectively"

Fig. 8

Observation of F-actin cytoskeleton with GFP fluorescence and peroxisomes with DsRED fluorescence mixed with the cell wall staining agent CFW in the FV-LifeAct-GFP and FV-DsRED-PTS1 strains, respectively"

Fig. 9

Observation of the infection structure formed by F. verticillioides conidia inoculated on maize silk and the circular assembly of F-actin proteins in the penetration structure"

Fig. 10

The F-actin polymerization inhibitor Latruculin A reduced the penetration ability of F. verticillioides on cellophane membrane"

Fig. 11

The effects of tricyclazole, CFW, and DPI on the penetration ability of F. verticillioides on cellophane membrane A control without drug treatment"

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